Drug resistance to HIV is a major threat to achieving long-term viral suppression in HIV+ individuals. Up to 16% of newly infected individuals acquire HIV with resistance to at least one of the major antiretroviral classes, and incomplete viral suppression and virologic failure are often associated with drug resistance. Therefore, current DHHS guideline recommends drug resistance testing before beginning or changing antiretroviral therapy. Genotypic assay based on population or bulk sequencing is the most commonly used method to determine HIV drug resistance mutations. However, because HIV circulates as quasispecies in vivo, current commercial assays are not sensitive in detecting minority drug resistant variants, which are known to compromise clinical response to antiretroviral therapy. Therefore, an accurate and sensitive assay that is capable of detecting drug resistant minority populations is urgently needed to determine the impact of HIV minor variants on viral suppression and guide rational selection of optimal antiretroviral therapy. The objective of this Phase I STTR application is to develop a sensitive assay that can accurately quantify HIV-1 minority variants. Recently, Dr. Wang's (PI, University of Florida) laboratory developed a Single Variant Sequencing (SVS) approach, which takes advantage of the speed and accuracy of the high-throughput MiSeq technology, and a random sequencing tags strategy that removes biases and technical artifacts known to obscure true representations of minority variants. By developing a bioinformatics pipeline that automates analysis of drug resistance calls, his laboratory has applied this SVS approach to quantify minor HCV resistance-associated variants in clinical samples. Medosome Biotec and its research partner at UF hypothesize that this SVS approach can be optimized to quantify minority populations of drug resistant HIV-1 accurately. In Phase I, the team will test this hypothesis by pursuing two Specific Aims: 1) Optimize the SVS method for sensitive and accurate quantification of HIV-1 drug resistance minority variants, and 2) Conduct initial testing of the optimized SVS method using laboratory HIV strains and clinical isolates. The SVS approach for HIV resistance testing is innovative because it will leverage the speed, accuracy, and the long paired-end read capability of MiSeq personal sequencer, and the random sequencing tag method to produce a new HIV-1 resistance assay for quantitative detection of HIV-1 minor variants. If successful, the SVS assay will be validated and developed in Phase II studies. An accurate and sensitive low cost SVS assay will have tremendous commercialization potential, given the global burden of HIV with more than 35 million HIV+ individuals requiring resistance testing at least once.